This invention is generally in the field of methods for the treatment and prevention of inflammatory responses using peptides derived from selectins.
The adherence of platelets and leukocytes to vascular surfaces is a critical component of the inflammatory response, and is part of a complex series of reactions involving the simultaneous and interrelated activation of the complement, coagulation, and immune systems.
Endothelium exposed to "rapid" activators such as thrombin and histamine becomes adhesive for neutrophils within two to ten minutes, while endothelium exposed to cytokines such as tumor necrosis factor and interleukin-1 becomes adhesive after one to six hours. The rapid endothelial-dependent leukocyte adhesion has been associated with expression of the lipid mediator platelet activating factor (PAF) on the cell surface, and the appearance of other endothelial and leukocyte surface receptors.
The selectins are three structurally related membrane glycoproteins that participate in leukocyte adhesion to vascular endothelium and platelets, as reviewed by McEver Thromb. Haemostas. 66:80-87 (1991). P-selectin (CD62), previously known as GMP-140 or PADGEM protein, is a receptor for neutrophils and monocytes that is rapidly translocated from secretory granule membranes to the plasma membranes of activated platelets, as reported by Larsen, et al., in Cell 59, 305-312 (October 1989) and Hamburger and McEver, Blood 75:550-554 (1990), and endothelial cells, as reported by Geng, et al., Nature 343:757-760 (1990) and Lorant, et al., J. Cell Biol. 115:223-234 (1991). E-selectin (ELAM-1) is a cytokine-inducible endothelial cell receptor for neutrophils, as reported by Bevilacqua Proc. Natl. Acad. Sci. USA 84:9238-9242 (1987), monocytes, as reported by Hession, et al., Proc. Natl. Acad. Sci. USA 87:1673-1677 (1990), and memory T cells, as reported by Picker, et al. Nature 349:796-799 (1991) and Shimizu, et al. Nature 349:799-802 (1991). L-selectin (LAM-1, LECAM-1, Leu-8/Mel 14/TQ1 antigen, lymphocyte homing receptor), a protein expressed on myeloid cells and most lymphocytes, participates in neutrophil extravasation into inflammatory sites and homing of lymphocytes to peripheral lymph nodes, as reported by Laskey, et al., Cell 56:1045-1055 (1989), Siegelman, et al., Science 243:1165-1172 (1989); Kishimoto, et al., Science 245:1238-1241 (1989); Watson, et al., Nature 349-164-167 (1991).
The slower cytokine-inducible endothelial adhesion for leukocytes is mediated, at least in part, by E-selectin (ELAM-1), that is synthesized by endothelial cells after exposure to cytokines and then transported to the cell surface, where it binds neutrophils. The isolation, characterization and cloning of ELAM-1 is reviewed by Bevilacqua, et al., in Science 243, 1160-1165 (1989). L-selectin was characterized and cloned as reported by Lasky, et al., Cell 56, 1045-1055 (1989) (mouse) and Tedder, et al., J. Exp. Med. 170, 123-133 (1989). P-selectin (GMP-140) was first purified from human platelets by McEver and Martin, J. Biol. Chem. 259:9799-9804 (1984). The protein is present in alpha granules of resting platelets but is rapidly redistributed to the plasma membrane following platelet activation, as reported by Stenberg, et al., (1985). The presence of P-selectin in endothelial cells and its biosynthesis by these cells was reported by McEver, et al., Blood 70(5) Suppl. 1:355a, Abstract No. 1274 (1987).
Proteins involved in the hemostatic and inflammatory pathways are of interest for diagnostic purposes and treatment of human disorders. However, there are many problems using proteins therapeutically. Proteins are usually expensive to produce in quantities sufficient for administration to a patient. Moreover, there can be a reaction against the protein after it has been administered more than once to the patient. It is therefore desirable to develop peptides having the same, or better, activity as the protein, which are inexpensive to synthesize, reproducible and relatively innocuous.
The function of GMP-140 (P-selectin) for use in inhibiting an inflammatory response was described by McEver in U.S. Ser. No. 07/320,408, filed Mar. 8, 1989, now U.S. Pat. No. 5,378,464. Peptides derived from GMP-140 are described in the continuation-in-part application U.S. Ser. No. 07/554,199, now abandoned, entitled "Functionally Active Selectin-Derived Peptides" filed Jul. 17, 1990 by Rodger P. McEver that are useful in diagnostics and in modulating the hemostatic and inflammatory responses in a patient wherein a therapeutically effective amount of a peptide capable of blocking leukocyte recognition of GMP-140 (P-selectin) is administered to the patient. U.S. Ser. No. 07/554,199 filed Jul. 17, 1990, now abandoned, also discloses that peptide sequences within the lectin domain of GMP-140, having homology with the lectin domains of other proteins, especially ELAM-1 (E-selectin) and the homing receptor (L-selectin), selectively inhibit neutrophil adhesion to purified GMP-140, and can therefore be used in diagnostic assays of patients and diseases characterized by altered binding by these molecules, in screening assays for compounds altering this binding, and in clinical applications to inhibit or modulate interactions of leukocytes with platelets or endothelial cells involving coagulation and/or inflammatory processes.
U.S. Ser. No. 07/757,131, now abandoned, entitled "Peptide Inhibitors of Inflammation Mediated by Selectins" filed Sep. 10, 1991 by George A. Heavner, Rodger P. McEver, and Jian-Guo Geng, and U.S. Ser. No. 07/699,693 filed May 14, 1991, now abandoned, disclose synthetic peptides derived from the core regions of GMP-140, residues 56-60 and residues 23-30, respectively. These peptides are useful in inhibiting binding of the selectins. It is preferable to develop peptides which can be prepared synthetically, having activity at least equal to, or greater than, the peptides derived from the protein itself.
It is therefore an object of the present invention to provide peptides interacting with cells recognized by selectins, including P-selectin, E-selectin and L-selectin.
It is another object of the present invention to provide methods for using these peptides to inhibit leukocyte adhesion to endothelium or to platelets.
It is a further object of the present invention to provide methods for using these peptides to modulate the immune response and the hemostatic pathway.
It is yet another object of the present invention to provide peptides for use in diagnostic assays relating to P-selectin, E-selectin and L-selectin.